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Suspension-feeding bivalve molluscs perform important ecological roles by coupling pelagic and benthic systems during their feeding activities. Particle capture, and thus feeding, is dependent on particle encounter and retention on the gill filaments, with several factors influencing this process. Over the past 30 yr, different types of synthetic microspheres have been used to examine aspects of particle capture and ingestion by bivalves. Critics of this work have posited that manufactured particles may contain surfactants, chemicals commonly used in manufacturing to reduce surface tension, that could produce spurious capture and ingestion rates. The goal of this work was to experimentally assess whether the presence of different types of surfactants on manufactured polystyrene particles can result in instantaneous effects on particle capture by the blue mussel Mytilus edulis. The effects of 3 different types of common surfactants (sodium dodecyl sulfate, benzalkonium chloride, Triton-X) on clearance rates (CR) and capture efficiencies (CE) were tested. Results indicated that none of the surfactant treatments had an effect on CR. Treatment with one of the surfactants (Triton-X) significantly lowered CE for 3 µm sized spheres compared to the control spheres (Milli-Q treated). None of the other tested surfactants significantly affected CE when compared to the control treatment. These data add to an understanding of particle handling by bivalves, and suggest that concentrations of surfactants found on commercially available microspheres used for experiments or found in the environment have little immediate effect on feeding processes.

Bivalves have ecological and economic importance but information regarding their associated microbiomes is lacking. As suspension feeders, bivalves capture and ingest a myriad of particles, and their digestive organs have a high throughput of particle-associated microbiota. To better understand the complement of transient and resident microbial communities, standard methods need to be developed. For example, fecal sampling could represent a convenient proxy for the gut microbiome and is simple, nondestructive, and allows for sampling of individuals through time. The goal of this study was to evaluate fecal sampling as a reliable proxy for gut microbiome assessment in the blue mussel (Mytilus edulis). Mussels were collected from the natural environment and placed into individual sterilized microcosms for 6 h to allow for fecal egestion. Feces and gut homogenates from the same individuals were sampled and subjected to 16S rRNA gene amplicon sequencing. Fecal communities of different mussels resembled each other but did not resemble gut communities. Fecal communities were significantly more diverse, in terms of amplicon sequence variant (ASV) richness and evenness, than gut communities. Results suggested a mostly transient nature for fecal microbiota. Nonetheless, mussels retained a distinct resident microbial community in their gut after fecal egestion that was dominated by ASVs belonging to Mycoplasma. The use of fecal sampling as a nondestructive substitute for direct sampling of the gut is strongly discouraged. Experiments that aim to study solely resident bivalve gut microbiota should employ an egestion period prior to gut sampling to allow time for voidance of transient microbes.

Suspension-feeding bivalve molluscs have evolved a highly effective mechanism for particle discrimination, which allows them to process efficiently the material to which they are exposed. The mechanisms controlling this preingestive sorting process have been described as either passive or active. Evidence of a passive selection mechanism has been demonstrated in several species of suspension-feeding bivalves. In contrast, to date there has been no evidence that active selection mechanisms underlay particle selection in bivalves, although the possibility of such mechanisms has been hypothesized numerous times. The present study was designed to examine active selection on the gill of the eastern oyster Crassostrea virginica. The gill was chosen for study because in oysters this organ is involved in particle selection. Two in vivo assays were designed in which the gill was exposed to dissolved cell exudate or extracts from the microalga Tetraselmis chuii, and the transport of polystyrene microspheres (25 µm) by the frontal cilia of the ordinary filament quantified by means of video endoscopy. Results demonstrated that the addition of exudates or extracts of T. chuii cells had no significant effect on the percentage of particles being transported dorsally (likely ingested) or ventrally (more likely rejected), and no differences in the number of particles in the ventral groove between control and experimental treatments. Results of follow-up experiments using covalently bound neoglycoproteins commonly found on cell surfaces of microalgal species demonstrated differences in transport of captured microspheres (10 µm, carboxylated) depending on the sugar type. Although chemoreception cannot be completely ruled out, these findings further indicate that physicochemical properties of particles, and not an active behavioral or physiological response (i.e., chemoreception of dissolved metabolites) by the animal, mediate particle selection in oysters.

In aquatic environments, suspension-feeding bivalve molluscs are exposed to a manifold of natural and anthropogenically derived particles, including micro- and nanoplastics. Plastic particles interact with feeding and digestive organs and can produce negative effects. As a result of these effects and the potential transfer of microplastics to higher trophic levels, including humans, there has been renewed interest in the ingestion of plastic particles by different species of bivalves. Many recent studies, however, have ignored the ability of bivalves to select among particles both pre- and post-ingestively. Neglecting to consider the factors that mediate particle capture, ingestion, and egestion can lead to erroneous data and conclusions. This paper outlines the current state of knowledge of particle processing by bivalves, and demonstrates how it relates to studies utilizing plastic particles. In particular, the effects of particle size, shape, and surface properties on capture, preferential ingestion, post-ingestive sorting, and egestion are summarized. The implications of particle selection for the use of bivalves as bioindicators of microplastic pollution in the environment are discussed. Only through a full understanding of the types of plastic particles ingested and egested by bivalves can internal exposure, toxic effects, and trophic transfer of microplastics be assessed adequately.

Given their particle feeding behavior, sessile nature, and abundance in coastal zones, bivalves are at significant risk for exposure to oil and oil dispersant following environmental disasters like the Deepwater Horizon oil spill. However, the effects of oil combined with oil dispersants on the health of oysters are not well studied. Therefore, eastern oysters (Crassostrea virginica) were exposed in vivo to Corexit® 9500, crude oil (high-energy water accommodated fraction; HEWAF), and a Corexit®/oil mixture (chemically-enhanced water accommodated fraction; CEWAF) to evaluate potential toxic effects on immunological (phagocytosis and respiratory burst), physiological (feeding rate), and histological endpoints. Phagocytosis was significantly increased following CEWAF exposure only. Respiratory burst was significantly decreased following Corexit® exposure, but significantly increased following exposure to the highest concentration of CEWAF. Oyster feeding rates were significantly decreased following exposure to Corexit®, HEWAF, and CEWAF, and were most sensitive to CEWAF exposure. These modulations of important immunological and physiological functions could result in serious health outcomes for oysters, such as increased parasitism and decreased growth. Our experiments showed that subtle, sub-lethal effects occurred following acute in vivo exposure to Corexit®, HEWAF, and CEWAF, though oysters were not equally sensitive to the three components. Data from this study can be used for more accurate risk assessment concerning the impact of oil and Corexit® on the health of oysters.

Rapid improvements in the technology used to assess microbial communities have led to an expansion of the breadth and scope of microbial ecology research over the past 20 y. The rapid increase in microbiome research, spurred by nextgeneration amplicon sequencing, has allowed researchers to characterize the microbial communities of organisms and environments which were previously poorly understood. Research on marine invertebrates is still developing, with the mechanisms that determine interactions between the host and their associated microbes largely unknown. Bivalves are of particular interest because of their crucial ecological and economic roles. With the anticipation of shifting environmental conditions, defining the natural variation and role of bivalve-associated microbes is vital to more complex research questions. The current knowledge of the spatial and temporal distributions of bivalve-associated microbes, microbial functional and genetic diversity, host-specific and tissue-specific interactions, and core microbial community composition is summarized here, as well as the beginning stages of understanding the role of microbiome in bivalve physiology and disease susceptibility.

Suspension-feeding bivalve molluscs are foundation species in coastal intertidal systems. The selective feeding capabilities of these animals can have a large influence on phytoplankton communities and nutrient flow to the benthos. Particle selection, including the types of particles chosen for ingestion and the possible mechanisms mediating selection, has been studied extensively and reported in the literature. To date, however, the possible mechanisms mediating these selective processes have remained elusive. Generally, the focus on a few key commercial species, and their demonstrated range of selective capabilities, has made it difficult to design studies that elucidate the mechanisms behind particle selection. This review focuses on key research that has been carried out in the last 20 y toward better understanding the mechanism that underlays selective capture and ingestion of particles in this important group of animals. Recently, work has been completed which has advanced the field in pointing to a passive mechanism as a mediator of selection, with the interactions between the physicochemical properties of particles and the mucus covering the pallial organs most likely mediating food choice. Although no strong evidence for an immediate, active mechanism which underlies particle selection was found, avenues for future research are suggested in this review. The possible mechanisms that control capture, including qualitative precapture selection, are also summarized and discussed in depth. Methodological considerations for rigorous experiments to advance the field are also discussed, including suggestions of general guidelines for experimental designs, which will allow better comparison of findings across studies.

The response of oyster (Crassostrea virginica) hemocytes was studied following exposure to anatase nanoparticles (ca. 7.4nm), surface-coated rutile nanocomposites (UV-Titan M212, ca. 86nm) and bulk titanium dioxide (TiO2) particles (anatase and rutile crystalline forms; 0.4-0.5μm). Hemocytes were collected from oysters and exposed to one of the four particle types at concentrations of 0.1, 0.5, and 1.0mg/L under dark and environmentally-relevant light conditions for periods of two and four hours. Hemocyte mortality, phagocytosis, and reactive oxygen species (ROS) production were then evaluated using flow-cytometric assays. Bulk and nanoparticulate TiO2 had little effect on viability of oyster hemocytes or on production of ROS. Significant changes in phagocytosis occurred after exposure to anatase nanoparticles for 4h under dark conditions, and UV-Titan for 2h under light conditions. Results demonstrate that TiO2 particles (bulk or nanoscale) produce minimal effects on hemocyte biomarkers examined following acute, in vitro exposures.

The capabilities of suspension-feeding bivalve molluscs to discriminately ingest particles are well known. Physicochemical properties of particles have been shown to play a role in mediating post-capture selection in these animals. In particular, particle surface charge and wettability, a proxy for hydrophobic-hydrophilic forces, can be used by different bivalve species as determinants of selection. How physicochemical factors of particles affect their capture, however, have been little studied. To investigate such interactions, a series of adhesion assays and particle capture experiments were designed. First, mucus from the gills of the blue mussel, Mytilus edulis (Linnaeus 1758), and the bay scallop, Argopecten irradians (Lamarck 1819), were isolated separately, adsorbed onto glass microscope slides, and incubated with polystyrene microspheres (10 μm) that were either untreated or covalently bound to different neoglycoproteins (NGP; N-acetyl-glucosamine, D-mannose), or bovine serum albumin (BS...

Nanoparticles are entering natural systems through product usage, industrial waste and post-consumer material degradation. As the production of nanoparticles is expected to increase in the next decade, so too are predicted environmental loads. Engineered metal-oxide nanomaterials, such as titanium dioxide, are known for their photocatalytic capabilities. When these nanoparticles are exposed to ultraviolet radiation in the environment, however, they can produce radicals that are harmful to aquatic organisms. There have been a number of studies that have reported the toxicity of titanium dioxide nanoparticles in the absence of light. An increasing number of studies are assessing the interactive effects of nanoparticles and ultraviolet light. However, most of these studies neglect environmentally-relevant experimental conditions. For example, researchers are using nanoparticle concentrations and light intensities that are too high for natural systems, and are ignoring water constituents that can alter the light field. The purpose of this review is to summarize the current knowledge of the photocatalytic effects of TiO2 nanoparticles on aquatic organisms, discuss the limitations of these studies, and outline environmentally-relevant factors that need to be considered in future experiments.

Microplastics (MP; 1 μm to 1 mm) of various shapes and compositions are ingested by numerous marine animals. Recently, proposals have been made to adopt bivalve molluscs as bioindicators of MP pollution. To serve as indicators of MP pollution, however, the proposed organisms should ingest, without bias, the majority of plastic particles to which they are exposed. To test this premise, eastern oysters

The capabilities of bivalve molluscs to feed selectively have been well documented, and physicochemical properties of particles have been implicated as possible factors in the selection process. In this study, the surface-property profiles of nine different microalgal species were determined by characterizing the surface charge, wettability (= contact angle), and surface carbohydrate moieties. Three fluorescein isothiocyanate (FITC) conjugated lectins were used to characterize carbohydrate moieties, including concanavalin A (ConA), Pisum sativum agglutinin (PEA), and wheat germ agglutinin (WGA). Distinct surface-property profiles were identified using linear discriminant analysis (DA) and used to design mixed-algal feeding experiments to assess particle selection by the blue mussel Mytilus edulis and the eastern oyster Crassostrea virginica. Results demonstrated preferential ingestion of some algal species over others, with strong rejection of some species (e.g. Pavlova lutheri and Prasinocladus marinus). These data were then used to develop DA and multiple linear regression models that considered the quantified surface properties and microalgal fates (rejected, ingested, or no selection) to examine determinants of selection. The DA model correctly classified 58% of the selection outcomes in mussels and 57% in oysters. Wettability was the most important factor in predicting selection in mussels, and surface charge was most important for oysters. In the multiple linear regression analyses, lectin affinity and wettability were the strongest predictors of particle selection, explaining ca. 90% of the variability in electivity index for mussels and 94% of the variability for oysters. By characterizing both physical and chemical surface properties of several microalgaeused as food for suspension-feeding bivalves, we demonstrate that multiple surface-property characteristics need to be considered in order to develop meaningful models of particle selection in bivalves. Future research also should take into account species-specific differences in selection.

Marine snow is a predominant form of sinking particulate carbon in the marine water column and represents a mechanism for transporting microplastics to the sea floor. We present a new dual density separation method employing sodium iodide extraction followed by methanol precipitation, specifically designed for microplastic isolation and identification in natural marine snow samples. A total of 59 microscopic particles from eight marine snow samples collected at Avery Point, CT were confirmed as plastics and/or substances containing typical plastic manufacturing additives. Extraction efficiency of this method was determined using polyethylene microspheres of varying sizes (63–75 μm, 212–250 μm and 500–600 μm) yielding 90%, 93% and 98% recoveries, respectively. Residual organic matter which can cause interference in downstream Raman spectroscopic analyses was eliminated by employing a 15% hydrogen peroxide (H2O2) digestion step, which caused negligible chemical modifications to the polymer samples. Extensive precautions such as combusted glassware, a microfiltration air hood, and incorporation of process blank samples ensured that airborne microplastic contamination was avoided. A phase contrast microscope equipped with a Raman spectrophotometer system using a 785 nm laser excitation source efficiently identified anthropogenic polymer materials. Unexpectedly, plastic additives such as pigments complicated the identification of polymers but their spectra were successfully interpreted through spectral subtraction and comparison to a database and authentic standards. The protocol described can be applied to detect microplastic in marine snow samples and improve our understanding of the fate of microplastic in the ocean.

Titanium dioxide nanoparticles (n-TiO2) are emerging contaminants and the ecological impact of these materials to the nearshore environment is largely unknown. The reactivity of n-TiO2 increases with light exposure, and the photocatalytic effects have been shown on cultures of bacteria and microalgae in the laboratory. The purpose of this study was to assess the response of natural bacterial and microalgal communities associated with marine aggregates to n-TiO2 under conditions similar to those found in the photic zone of nearshore waters. Nano and bulk TiO2 particles were incorporated into marine aggregates over 4 days under two light conditions: 6:18 and 0:24 (hours light:dark). The abundance and metabolic response of heterotrophic bacteria and viability of microalgae associated with aggregates were assessed. Although the proportion of living microalgae was unchanged, the abundance, total metabolic activity and functional diversity of heterotrophic bacteria were significantly altered by irradiated n-TiO2.

Marine aggregates incorporate particles from the environment, including microplastic (MP). The characteristics of MP in aggregates and the role of aggregates in linking MP with marine organisms, however, are poorly understood. To address these issues, we collected aggregates and blue mussels, Mytulis edulis, at Avery Point, CT, and analyzed samples with microspectrometers. Results indicate that over 70% of aggregates sampled harbored MP (1290 ± 1510 particles/m3). Fifteen polymer types were identified, with polypropylene, polyester and synthetic-cellulose accounting for 44.7%, 21.2% and 10.6%, respectively, of the total MP count. Over 90% of MP in aggregates were ≤1000 μm, suggesting that aggregations are a sink for this size fraction. Although size, shape, and chemical type of MP captured by mussels were representative of those found in aggregates, differences in the sizes of MP in pseudofeces, feces and digestive gland/gut were found, suggesting size-dependent particle ingestion. Over 40% of the MP particles were either rejected in pseudofeces or egested in feces. Our results are the first to identify a connection between field-collected marine aggregates and bivalves, and indicate that aggregates may play an important role in removing MP from the ocean surface and facilitating their transfer to marine food webs.

Given their particle feeding behavior, sessile nature, and abundance in coastal zones, bivalves are at significant risk for exposure to oil and oil dispersant following environmental disasters like the Deepwater Horizon oil spill. However, the effects of oil combined with oil dispersants on the health of oysters are not well studied. Therefore, eastern oysters (Crassostrea virginica) were exposed in vivo to Corexit

In the marine environment, settling particulates have been widely studied for their role as effective vertical transporters of nutrients and metals scavenged from the euphotic zone to the benthos. These particulates are composed of transparent exopolymers, plankton and bacterial cells, detritus and organic matter, and form various size fractions from colloids (300 μm). As marine snow forms in the water column, anoxic layers form around and within the aggregation potentially creating a prime environment for the methylation of mercury (Hg), which occurs primarily in low oxygen environments. To examine this process, marine aggregates were produced from sieved estuarine seawater (100 μm) in 1-L glass bottles spiked with stable isotope enriched methylmercury (CH3199Hg) and inorganic mercury (200Hg(II)) at 18° C using a roller-table. After the rolling period, different particle-size fractions were collected and analyzed, including: visible marine snow (>300μm), particulates 8 to 300 μm, and particulates 0.2 to 8μm. Particulate analysis indicated higher incorporation of both forms of Hg into marine snow compared to unrolled treatments, with greater incorporation of 200Hg(II) than CH3199Hg. In addition, inorganic Hg was methylated and CH3Hg was demethylated in the larger particulate fractions (>8μm). Methylation and demethylation rates were assessed based on changes in isotopic composition of Hg(II) and CH3Hg, and found to be comparable to methylation rates found in sediments. These results indicate that net Hg methylation can occur in marine snow and smaller aggregates in oxic coastal waters, and that this net formation of CH3Hg may be an important source of CH3Hg in both coastal and open ocean surface environments.

Historically, particle capture efficiency (CE) in suspension-feeding bivalve molluscs has been shown to be strongly dependent on particle size, increasing asymptotically to a maximum of about 100% for particles ca. ≥ 4 μm in diameter. Recent advances in the analysis of the particulate matter of seston have allowed for more precise studies of bivalve feeding under natural conditions. Some studies have reported that the mechanisms associated with particle capture exhibit physiological plasticity, and under certain conditions smaller cells and particles are captured in preference to larger ones. For bivalves, however, there is no mechanistic explanation that would account for such fine-scale control of CE based on size. The current study experimentally assessed the seasonal control of CE by the blue mussel, Mytilus edulis , employing a flow-through system to examine particle capture of natural seston. The natural particle field was analyzed using two different types of particle analyzers, the LISST-100X and the Coulter Multisizer IIe. Mussels were simultaneously delivered synthetic microspheres of defined diameter (2–45 μm) to control for the effects of seasonal differences in the size and shape of natural particles. The capture of microspheres was quantified by means of flow cytometry (FCM), and results cross-checked with the Multisizer. Additionally, gene expression of a mucosal lectin (MeML) associated with the feeding organs of mussels was examined as a biomarker for physiological response to seasonal changes in the particle-food supply. Results demonstrated that for microspheres ≥ 4 μm CE of mussels was always near 100%, and did not change seasonally. In contrast, there was an apparent seasonal shift in CE of natural particles, with particles 17-to-35 μm in equivalent spherical diameter (ESD) occasionally being captured at lower efficiencies than particles 4-to-15 μm in ESD (e.g., during September and December). No relationship between MeML expression and seasonal CE was found. These findings call into question the physiological plasticity of CE in mussels and alternative hypotheses are presented. We suggest that the purported changes in CE are not a consequence of behavioral or physiological responses of mussels, but rather a result of one or more of the following confounding factors; 1) instrument artifacts that can arise as a result of the way in which laser and electronic particle counters calculate ESD to estimate particle size; 2) disaggregation of flocculent material collected from control chambers; 3) post-capture escape of highly motile phytoplankton cells from the infrabranchial camber; 4) qualitative factors of the particles that could affect capture; or 5) mathematical happenstance of calculating CE on particle-size classes that contain widely different numbers of particles.

The production rates of titanium dioxide (TiO2) nanoparticles for consumer products far exceed the pace at which research can determine the effects of these particles in the natural environment. Sedentary organisms such as suspension-feeding bivalves are particularly vulnerable to anthropogenic contaminants, such as nanoparticles, that enter coastal environments. The purpose of this work was to examine the ingestion, bioaccumulation, and depuration rates of TiO2 nanoparticles by two species of suspension-feeding bivalves, the blue mussel (Mytilus edulis) and the eastern oyster (Crassostrea virginica). Two representative TiO2 nanoparticles, UV-Titan M212 (Titan) and Aeroxide P25 (P25), were delivered to the animals either incorporated into marine snow or added directly to seawater at a concentration of 1.0 mg/L for exposure periods of 2 and 6 h. After feeding, the animals were transferred to filtered-seawater and allowed to depurate. Feces and tissues were collected at 0, 12, 24, 72, and 120 h, post-exposure, and analyzed for concentrations of titanium by inductively coupled plasma-mass spectrometry. Results indicated that the capture and ingestion (i.e., transfer to the gut) of TiO2 nanoparticles by both mussels and oysters was not dependent on the presence of marine snow, and weight-standardized clearance rates of bivalves exposed to TiO2 nanoparticles were not significantly different than those of unexposed control animals. Both species ingested about half of the nanoparticles to which they were exposed, and >90% of the nanoparticles were egested in feces within 12 h, post-exposure. The findings of this study demonstrate that mussels and oysters can readily ingest both Titan and P25 nanoparticles regardless of the form in which they are encountered, but depurate these materials over a short period of time. Importantly, bioaccumulation of Titan and P25 nanoparticles does not occur in mussels and oysters following exposures of up to 6 h.

The microbial communities and overall health of the eastern oyster, Crassostrea virginica, have long been topics of interest due to the fundamental economic and ecological roles this species maintains. A broad scale characterization of the oyster microbiome over spatial and seasonal scales, however, has never been carried out. The primary goal of this study was to examine the factors mediating microbial communities of the gut and pallial fluid of C. virginica at three sites within the Long Island Sound estuary, with a focus on both genetic structure (T-RFLP) and physiological profiling (EcoPlates) of the microbiome. Results indicated that the genetic structure of microbial communities of oysters was minimally separated across sites, but was influenced by season. Although the microbial community structure was similar, the number of carbon sources utilized by these communities (richness) varied across site, season, and anatomical location within the host. Parameters including oyster condition index, Dermo disease, and ambient water temperature were measured to assess their influence on the oyster microbiome. Only water temperature was found to have a significant relationship with microbial community structure and richness. Results suggest that a core microbiome may exist within the eastern oyster, specifically for those populations that are not genetically distinct.

Nanoparticles have applications in a diverse range of products including medications, detergents, cosmetics, paint, sunscreen and electronics, with an economic worth projected to reach $2.5 trillion dollars in 2015. Research into the effects of manufactured nanomaterials on the environment, however, has failed to keep pace with the high volume of commercial production. Whereas a number of studies have examined the effects of nanoparticles on aquatic species, little work has focused on the way in which benthic marine species encounter, ingest and depurate these materials. The purpose of this study was to examine the ingestion and depuration of titania nanoparticles (anatase) by the blue mussel (Mytilus edulis) and the eastern oyster (Crassostrea virginica) during a spill scenario (an acute exposure to elevated concentrations). Bivalves were exposed to nanoparticles either incorporated into marine snow, an environmentally relevant medium for pollutants, or added directly to seawater at a concentration of 4.5 mg L−1 for 2 h. After feeding, the animals were transferred to filtered seawater and allowed to depurate. Faeces and tissues were collected at 0, 6, 24, 72 and 120 h, post-exposure, and analysed for concentrations of titanium by inductively coupled plasma-mass spectrometry. Results indicated that the capture and ingestion of titania nanoparticles by both species was not dependent on the method of delivery (incorporated into marine snow or freely suspended). Additionally, greater than 90% of the titania nanoparticles, on average, were eliminated from the tissues after 6 h, and only trace amounts remained after 72 h. These data demonstrate that mussels and oysters readily ingest titania nanoparticles, but rapidly depurate the material within hours of an acute exposure suggesting that little would be transferred to secondary consumers including humans. Further research is required to determine if other species of suspension-feeders handle titania nanoparticles in a manner similar to bivalves.

The response of oyster (Crassostrea virginica) hemocytes was studied following exposure to anatase nanoparticles (ca. 7.4nm), surface-coated rutile nanocomposites (UV-Titan M212, ca. 86nm) and bulk titanium dioxide (TiO

Observations are presented to elucidate the complex and highly efficient feeding mechanisms and habits in the common Atlantic slippersnail, Crepidula fornicata. Using a newly designed video system coupled with video endoscopy and microscopic observations, collection of food particles throughout the feeding cycle was documented. The process is described from postcapture particle transport on the gill, to production and consumption of the food cord, and the results were compared with controversial historical records and descriptions of feeding, especially with regard to the roles of the radula, food pouch, and a purported mucus net or filter. The food cord is produced continuously and is the dominant mode of feeding. Capture of food cords is efficient, but often sloppy; more than 10% of food volume production is ultimately expelled to the exterior of shell, making these energy-rich cords available to other benthic feeders. Although it was observed that the ciliated food pouch produces balls of mucu...

Biolog MicroPlates(TM) (e.g. EcoPlate(TM), MT2 MicroPlate(TM), GN MicroPlate(TM)) are useful tools for characterizing microbial communities, providing community-level physiological profiles to terrestrial and aquatic ecologists. The more recently designed Biolog EcoPlates have been used frequently in aquatic ecology with success. This study, however, reveals one major problem when using EcoPlates to evaluate samples within an estuarine or seawater matrix. At concentrations greater than 100 parts per million, the cation calcium begins to interfere with the microplate chemistry, causing false positive readings. Experiments, in which multiple treatments of natural and artificial seawater were tested, as well as calcium-addition experiments, demonstrate that calcium inhibits complete dissolution of the minimal growth medium in wells. Future studies involving Biolog EcoPlates and MicroPlates should take this effect into account, and the dilution of samples is strongly recommended to diminish the "calcium effect."

article i nfo Article history: The mechanisms by which bivalve molluscs differentiate among particles are not clearly understood. Recent evidence suggests that particle selection by bivalves can be mediated by interactions between carbohydrates attached to a particle's surface and lectins present in the mucus covering the feeding organs. The physico- chemical surface properties of particles have also been proposed as factors contributing to particle selection. In this study, we examined the effects of surface charge and wettability of 10-μm spheres on particle selection by two species of suspension-feeding bivalves: eastern oysters (Crassostrea virginica) and blue mussels (Mytilus edulis). Microspheres were delivered to bivalves in particle-selection assays, and the proportions of spheres rejected as pseudofeces and egested as feces were determined using flow cytometry. Results sug- gest that when given a choice, both mussels and oysters rejected some types of microspheres (e.g., aluminum oxide) and ingested other types (e.g., polystyrene). In some assays, microspheres with very different surface charges or wettabilities were handled similarly, indicating that neither property alone was a qualifier for selection. The differences in surface properties between pairs of microspheres were also considered and used as variables in discriminant analyses. For oysters and mussels, the generated models explained ca. 25% and 72% of the variation in the data, respectively. In both models, wettability was more important than charge in classifying data into their correct group (rejected, preferentially ingested, or not selected). Results from this study support the idea that non-specific physicochemical interactions can play a role in mediating selection in suspension-feeding bivalves.

While many coastal ecosystems previously supported dense meadows of seagrass and dense stocks of bivalves, the impacts of overfishing, eutrophication, harmful algal blooms, and habitat loss have contributed to the decline of these important resources. Anthropogenic nutrient loading and subsequent eutrophication has been identified by some researchers as a primary driver of these losses, but others have described potential positive effects of eutrophication on some estuarine resources. The Peconic Estuary, Long Island, NY, USA, offers a naturally occurring nutrient-loading gradient from eutrophic tidal creeks in its western reaches to mesotrophic bays in the eastern region. Over 2 years, we conducted an experiment across this gradient to examine the effects of eutrophication on the growth of estuarine species, including juvenile bivalves (northern quahogs (Mercenaria mercenaria), eastern oysters, (Crassostrea virginica), and bay scallops (Argopecten irradians)) and slipper limpet (Crepidula fornicata). Water quality and phytoplankton community biomass and composition were concurrently monitored at each site, and the effects of these variables on the growth of estuarine species were analyzed with multiple regression model. Eutrophication seemed to impact shellfish through changes in the quality of food and not the quantity since the growth rates of shellfish were more often correlated with densities of specific cell types or quality of seston rather than bulk measures of phytoplankton and organic seston. Northern quahogs and eastern oysters grew maximally within eutrophic locales, and their growth was positively correlated with high densities of autotrophic nanoflagellates and centric diatoms in these regions (p

Parasites have developed myriad strategies to reach and infect their specific hosts. One of the most common mechanisms for non-vector transmitted parasites to reach the internal host environment is by ingestion during feeding. In this study, we investigated the mechanisms of oyster host colonization by the alveolate Perkinsus marinus and focused on how oysters process infective waterborne P. marinus cells during feeding in order to determine the portal(s) of entry of this parasite to its host. We also compared the infectivity of freely-suspended cells of P. marinus with that of cells incorporated into marine aggregates to link changes in particle processing by the feeding organs with infection success and route. Finally, we evaluated the effect of oyster secretions (mucus) covering the feeding organs on P. marinus physiology because these host factors are involved in the processing of waterborne particles. The ensemble of results shows a unique mechanism for infection by which the parasite is mostly acquired during the feeding process, but not via ingestion. Rather, infection commonly occurs during the rejection of material as pseudofeces before reaching the mouth. The pseudofeces discharge area, a specialized area of the mantle where unwanted particles are accumulated for rejection as pseudofeces, showed significantly higher parasite loads than other host tissues including other parts of the mantle. Aggregated P. marinus cells caused significantly higher disease prevalence and infection intensities when compared to freely-suspended parasite cells. Mucus covering the mantle caused a quick and significant increase in parasite replication rates suggesting rapid impact on P. marinus physiology. A new model for P. marinus acquisition in oysters is proposed.

The in vivo response of adult northern qua- hogs, Mercenaria mercenaria ,t oAureococcus anophagef- ferens (brown tide) at the level of the gill was determined using video-endoscopy. Feeding activity, particle-approach velocities, and ventral-groove-transport velocities were documented after the quahogs were exposed to Isochryis galbana (baseline observations) supplemented with either toxic or nontoxic A. anophagefferens at two bloom concen- trations (8 10 5 or 2 10 6 cells ml -1 ). Externally, there was no evidence of adverse effects of brown tide on feeding, as siphons remained extended and dilated. Toxic brown tide at both concentrations elicited gill muscular contractions, intermittent cessation of water flow, and decreased particle loading within the pallial cavity. The 8 10 5 cell ml -1 toxic treatment had no significant effect on approach velocities or ventral-groove-transport velocities after 2 h, although time- averaging showed significant reduction of the latter during the last 30 min of exposure. The higher concentration of toxic brown tide caused a significant decrease in these velocities after only 1 h. Nontoxic brown tide produced none of these effects. Thus, A. anophagefferens compro- mised quahog feeding by stimulating contractions of the branchial musculature and interfering with lateral and ven- tral groove ciliary beating. These effects were both time- and concentration-dependent and could be caused by either a dopaminergic or a serotonergic toxic factor.

This study provides evidence that the suspen- sion-feeding blue mussel, Mytilus edulis, uses biochemical cues to recognize its food. We identified lectins in mucus from the gills and labial palps, two pallial organs involved in the feeding process. These compounds were able to agglutinate rabbit and horse erythrocytes (RBC) and several species of marine microalgae representing different fami- lies. Additionally, the agglutination of RBC and microalgae was inhibited by several carbohydrates (fetuin, lipopolysac- charide (LPS), and mannose-related residues), suggesting that a suite of lectins may be present in mucus from the gills and labial palps. Results from feeding experiments, using microspheres with tailored surfaces, demonstrated that mus- sels preferentially ingested microspheres coated with the neoglycoproteins glucosamide-BSA and mannopyranosyl- phenyl-BSA but rejected in pseudofeces microspheres coated with BSA alone. The positive selection for neogly- coprotein-coated microspheres was inhibited when mussels were pre-incubated in seawater containing a solution of the same neoglycoprotein. Two surface properties of the micro- spheres, charge and wettability, had little effect on the observed selection process. Our results, along with our previous findings for oysters, suggest a new concept for the mechanism of particle selection in bivalves and perhaps other suspension-feeding organisms. Specifically, the selec- tion of particles involves interactions between epiparticulate carbohydrates and lectins in the mucus produced by feeding organs.

Despite advances in the study of particle selection in suspension-feeding bivalves, the mechanisms upon which bivalves rely to discriminate among particles have not been elucidated. We hypothesized that particle sorting in suspension-feeding bivalves could be based, in part, on a biochemical recognition mechanism mediated by lectins within the mucus that covers the feeding organs. Using Crassostrea virginica, the Eastern oyster, our investigations demonstrated that lectins from oyster mucus can specifically bind several microalgal species as well as different types of red blood cells (RBC), triggering their agglutination. Agglutination of microalgal species and RBC varied with the source of mucus (gills vs. labial palps). Hemagglutination and hemagglutination inhibition assays emphasized that mucus contains several lectins. In feeding experiments, Nitzschia closterium and Tetraselmis maculata were separately incubated with mucus before being fed to oysters. Results showed that pre-treating these microalgae with mucus significantly alters the ability of oysters to sort particles. In another experiment, oysters were fed a mixture of microspheres coated with either bovine serum albumin (BSA) or glucosamide-BSA. Results show that oysters preferentially ingest microspheres with bound carbohydrates, highlighting probable interactions between lectins and carbohydrates in the mechanisms of microalgae recognition. This study confirms the presence of lectins in mucus that covers the feeding organs of oysters and suggests a new concept with regard to particle processing by suspension-feeding bivalves: specific interactions between carbohydrates on the surface of particles and lectins within the mucus mediate the selection and rejection processes.

The enzyme-labeled fluorescence (ELF-97®) substrate produces an insoluble, fluorescent yellow-green product at the site of alkaline-phosphatase (AP) activity. Fifteen coastal phytoplankton species were tested to determine if AP activity could be detected in phosphate- depleted media. All species tested, except Synechococcus bacillaris, expressed AP activity. Subsequently, threshold concentrations of soluble reactive phosphate (SRP) at which AP activity could be detected by ELF-97® were deter- mined for Chaetoceros neogracile, Chlorella autotrophica, Isochrysis sp., Prorocentrum minimum ,a ndTetraselmis chui. Microalgal species differed significantly in the SRP con- centration at which AP activity was first detectable (10.1- 16.4 µM), well above concentrations normally considered limiting for phytoplankton. P. minimum began to express AP activity at a higher SRP concentration than the other algal species; this may be attributable to a relatively high DNA/cell ratio in P. minimum, compared to the other phytoplankton. Thus, phytoplankton species may respond to phosphorus deficiency at high SRP concentrations.

Oysters accumulate relatively low levels of domoic acid (DA) compared to other bivalves. Mafra et al. (2009, in this Theme Section) identified feeding mechanisms of oysters that may lead to low DA accumulation during monospecific blooms of Pseudo-nitzschia multiseries. However, several differ- ent species of Pseudo-nitzschia, as well as other diatoms and flagellates, may co-occur during a bloom. Therefore, the present study investigates pre-ingestive feeding processes that operate when oysters Crassostrea virginica are exposed to mixed phytoplankton assemblages containing P. multiseries of varying cell length. Guided by video-endoscopy, material transported along the ventral and dorsal gill tracts was sampled and analyzed to determine the site for sorting of microalgae on the pallial organs. There was no preferential rejection of P. multiseries in pseudofeces when oysters were exposed to the alga in a mixed suspension with other diatom species (Thalassiosira weissflogii or Chaetoceros muelleri). In contrast, P. multiseries was preferentially rejected when mixed with the flagellates Iso- chrysis galbana or Rhodomonas lens, suggesting a qualitative mechanism for particle sorting. This occurred on the gills, followed by further selection on the palps. Oysters also preferentially rejected larger P. multiseries cells (82 to 90 µm) relative to smaller ones (24 to 28 µm) on the gills, while no fur- ther selection based on size occurred on the palps. This effect is attributed to the fact that P. multiseries cells with a length that exceeds the width of the principal filament aperture (ca. 68 µm) are more likely directed to the ventral tract and rejected in pseudofeces. These findings offer an additional explanation for the relatively low DA levels found in oysters during natural Pseudo-nitzschia spp. blooms.

Many studies on the biology of bivalves have focused on identifying the importance of various environmental factors in regulating feeding behaviour in order to predict ingestion and growth responses in a variety of habitats. When taking into consideration the diversity of studies undertaken and the variety of methodologies employed, it is perhaps not surprising that there have been inconsistencies in both interspecific and intraspecific comparisons. In order to compare and contrast the way in which feeding activities of a mussel species ( Mytilus edulis ) and a scallop species ( Placopecten magellanicus ) respond to environmental factors and seston characteristics, we exposed both species simultaneously to a fluctuating assemblage of natural particles found in their local environment. Similar sizes of mussels (40–73 mm) and scallops (40–88 mm) were held in flow through chambers at two different sites in Atlantic Canada on four separate occasions, during which environmental conditions, including seston characteristics, were monitored and individual clearance rates were measured. Because the same individuals were measured over time, repeated-measures statistical procedures were used to analyze the data. Results indicate that weight standardized clearance rates of mussels were significantly higher than those of scallops only at the highest concentration tested (12.8 mg l − 1 ), which translates into almost 3 times more material being filtered. Scallops appeared to respond to fluctuations in environment parameters and suspended food particles more consistently than mussels. These data suggest that the feeding behaviour of these two species is mediated differently by the same environmental parameters. Our study also demonstrates the importance of selecting appropriate statistical tests (e.g., repeated measures) for the analyses of data obtained from repeated sampling of the same individuals over time, and the need to consider species-specific feeding responses when developing models that predict the influence of bivalve feeding on ecosystem function.

Transparent exopolymer particles (TEP) are known to form from dissolved precursors released by phytoplankton and bacteria. Many benthic suspension feeders also have been shown to produce significant amounts of TEP under field and laboratory conditions. Although TEP from phyto- plankton have been implicated in enhancing aggregation and sedimentation of suspended material, the role of TEP from suspension feeders in aggregate formation is still unknown. In order to deter- mine if TEP produced by suspension feeders enhance the formation of marine aggregates, organic matter from actively feeding blue mussels Mytilus edulis and sea vases Ciona intestinalis held in recirculating chambers was collected. Control water was collected from chambers without animals. Animal-conditioned and control water were then mixed with 1 of 2 non-sticky particles (silica-amino beads or diatomaceous earth). The suspensions were transferred to 250 ml bottles, which were placed on a roller table to generate aggregates. Particle aggregation was quantified by examining the size distribution of particles over time by means of an electronic particle counter (particles between 6 and 90 μm), and by counting large (greater than ca. 100 μm) aggregates under a microscope at the end of the experiment. We found that significantly more aggregates formed in the animal-conditioned water than in control water after 48 h, for both species and both kinds of added particles. The number of large aggregates that formed was related to TEP concentration, and not related to total or dissolved organic carbon. Our results suggest that suspension feeders produce significant quantities of TEP that can enhance particle aggregation.

Planar (2 dimensional) particle image velocimetry (PIV) was compared against the established clearance rate method of estimating bulk flow through suspension-feeding organisms to evaluate PIV as a technique to estimate feeding activity. The study of ciliary pumps has relied on advances in technology, as it is difficult to access the pump without excessively disturbing the animal. We used PIV to examine feeding velocities in five species of suspension-feeding, benthic invertebrates including one ascidian (Styela clava) and four bivalves (Mytilus edulis, Crassostrea virginica, Argopecten irradians and Mercenaria mercenaria). In calibration experiments using pipes of varying diameters, we were able to resolve flow with498.5% accuracy. We extended the pipe model to study water processing in the mussel M. edulis. Both velocity and bulk flow through the mussel changed in predictable ways in relation to clearance rate (P50.05). Application of PIV to the study of suspension feeding will further our understanding of the behavior and physiology of economically- and ecologically-important organisms.

Suspension-feeding molluscs are important members of coastal communities and a large body of literature focuses on their feeding processes, including the efficiency of particle capture. Some molluscs, such as bivalves, capture individual picoplankton cells (0.2–2.0 μm) with a retention efficiency of less than 50%, leading to the assumption that such particles are not an important food resource. Picoplankton, however, are often concentrated in particle aggregates of much larger size. This study investigates the ability of suspension feeders to ingest picoplankton-size particles (0.2–2.0 μm) bound in marine aggregates. We fed clams (Mercenaria mercenaria), mussels (Mytilus edulis), oysters (Crassostrea virginica), scallops (Argopecten irradians) and slipper snails (Crepidula fornicata) 1.0- and 0.5-μm fluorescent particles (either polystyrene beads or bacteria) that were (1) dispersed in seawater, or (2) embedded within laboratory-made aggregates. Dispersed 10-μm beads were also delivered so that feeding activity could be determined. Ingested fluorescent particles were recovered in feces or isolated digestive glands and quantified. Results indicate that aggregates significantly enhance the ingestion of 1.0- and 0.5-μm beads by all species of bivalves, and enhance the ingestion of bacteria (greatest cell dimension ca. 0.6 μm) by all suspension feeders examined. Differences among species in their ability to ingest aggregates and picoplankton-size particles, however, were evident. Compared to mussels and clams, scallops and oysters ingested fewer aggregates with 1.0-μm beads or bacteria, and slipper snails ingested the most dispersed beads and bacteria. These differences may be a consequence of variations in gill structure and mechanisms of particle processing. Our data demonstrate that suspension feeders can ingest picoplankton-size particles that are embedded within aggregates, and suggest that such constituent particles may be an important food resource.

Marine aggregates were evaluated for their potential role in the ecology of aquatic pathogens using underwater video surveys coupled with direct collection of aggregates in modified settling cones. Six locations, two each in New York, Connecticut, and Massachusetts, were surveyed over 8 months to explore differences in the characteristics of aggregates found in habitats populated by clams (Mercenaria mercenaria) and oysters (Cras- sostrea virginica). Microaggregate ( 500 lm) concentrations, but peak concentrations of macroaggregates and microaggregates, mean size of particles, and volume fraction of aggregated material varied among the six shallow-water habitats. Concentrations (colony- forming units per ml) of total heterotrophic bacteria (THB) and total mesophilic pathogenic bacteria (MPB) from samples of aggregates were significantly different among the four locations bordering Long Island Sound (LIS). The highest concentrations and enrichment factors in aggregates were observed in August for THB and in June for MPB. Significant correlations were detected for salinity and the concentrations and enrichment factors of THB in aggregates and for the concentrations and percentages of MPB in seawater samples. Significant correlations were also detected for temperature and the concentrations of MPB in aggregates and the enrichment factors for THB and MPB (marginal significance). Bacterial species identified in association with aggregates included: Vibrio cholerea, V. parahaemolyticus, V. vulnificus, V. alginolyticus, Aeromonas hydrophila, Pseudomonas aeruginosa, Escherichia coli, and Mycobacteria sp. These results have important implications for the way in which aquatic pathogens are collected, quantified, and monitored for risk-based surveillance in shallow-water ecosystems.

The economically important marine bivalve mollusc, Mercenaria mercenaria, (commonly called a northern quahog or hard clam), has endured considerable mortalities caused by a thraustochytrid pathogen called Quahog Parasite X (QPX). Data on the percent prevalence of QPX infections were compiled from published reports along with our data to describe the epizootiology of QPX disease. QPX infections occurred in clams collected from both cultured beds and wild populations, but a higher percentage of QPX cases (76.5%) were from cultured clam beds. In addition, samples from cultured beds had a significantly higher prevalence (29.2 ± 27.2%) of QPX infections compared with samples from wild populations (9.6 ± 9.6%). The highest prevalence of QPX infections occurred in clams from samples with an intermediate size range (shell lengths 20-55 mm). QPX infections occurred in both male and female clams, but infection prevalence does not appear to be correlated with sex or sex ratios. The geographical range of QPX-related clam mortalities was Atlantic Canada to the Eastern Shore of Virginia, USA. Only marginally significant differences were detected between the prevalence of QPX at different locations. There were no latitudinal gradients in QPX prevalence or frequencies, suggesting local factors were important in determining its distribution. Although QPX infections occurred throughout the year, no seasonal trends in the prevalence or frequencies of QPX were discernable. This summary of information available on QPX disease highlights the need for more thorough data collection regarding factors believed to be associated with its presence and severity in hard clams.

We describe a novel sensor based on fiber optic technology for fine scale (∼0.1 mm) laboratory measurements of valve gape in bivalve molluscs. We have illustrated the operation of this sensor by measuring valve gape in conjunction with depletion rate assays in the Eastern oyster (Crassostrea virginica, Gmelin 1791). The sensor is capable of accurate and repeatable measurements, with few of the drawbacks of other valve gape sensing methods (e.g., levers, strain gauges, electromagnetic sensors). This sensor technology can be applied to other systems requiring the measurement of axial distances while immersed in seawater or other harsh environments.

Bivalves process large volumes of water, leading to their accumulation of bacteria, including potential human pathogens (e.g., vibrios). These bacteria are captured at low efficiencies when freely suspended in the water column, but they also attach to marine aggregates, which are captured with near 100% efficiency. For this reason, and because they are often enriched with heterotrophic bacteria, marine aggregates have been hypothesized to function as important transporters of bacteria into bivalves. The relative contribution of aggregates and unattached bacteria to the accumulation of these cells, however, is unknown. We developed an agent‐based model to simulate accumulation of vibrio‐type bacteria in oysters. Simulations were conducted over a realistic range of concentrations of bacteria and aggregates and incorporated the dependence of pseudofeces production on particulate matter. The model shows that the contribution of aggregate‐attached bacteria depends strongly on the unattached bacteria, which form the colonization pool for aggregates and are directly captured by the simulated oysters. The concentration of aggregates is also important, but its effect depends on the concentration of unattached bacteria. At high bacterial concentrations, aggregates contribute the majority of bacteria in the oysters. At low concentrations of unattached bacteria, aggregates have a neutral or even a slightly negative effect on bacterial accumulation. These results provide the first evidence suggesting that the concentration of aggregates could influence uptake of pathogenic bacteria in bivalves and show that the tendency of a bacterial species to remain attached to aggregates is a key factor for understanding species‐specific accumulation.

We evaluated marine aggregates as environmental reservoirs for a thraustochytrid pathogen, Quahog Parasite Unknown (QPX), of the northern quahog or hard clam, Mercenaria mercenaria. Positive results from in situ hybridization and denaturing gradient gel electrophoresis confirm the presence of QPX in marine aggregates collected from coastal embayments in Cape Cod, Massachusetts, where QPX outbreaks have occurred. In laboratory experiments, aggregates were observed and recorded by entering a quahogÂs pallial cavity, thereby delivering embedded particles from the water column to its benthic bivalve host. The occurrence of pathogen-laden aggregates in coastal areas experiencing repeated disease outbreaks suggests a means for the spread and survival of pathogens between epidemics and provides a specific target for environmental monitoring of those pathogens.